Main Conference - Day 3 (May 14)Thursday, 14 May 2026

Phosphorothioate diester linkages play a critical role in the efficacy of many RNA therapeutics. However, there are inherent diastereomeric complexities that these linkages possess ultimately presenting challenges for both manufacturing and analytical characterization. This presentation highlights how Codexis’ ECO Synthesis® Manufacturing Platform enables control over diastereomeric composition, facilitating the scalable production of high-quality siRNA.

Traditional solid phase peptide synthesis often involves the use of large volumes of a number of challenging solvents and reagents, including polar aprotic solvents, chlorinated solvents, and TFA. Efforts to remove these types of solvents and reagents has led to concerted efforts to investigate alternative platform strategies that both eliminate these chemicals, and also drive down synthesis cost and PMI. Additionally, the synthesis of peptides using purely liquid phase chemistries offers an option to designate more advanced sub-fragments of the peptide with favorable physical properties as potential registered starting materials.

This presentation will discuss: 1) Direct sequence mapping of mRNA using online partial T1 digests and 2D LC MS; 2) Multi-attribute monitoring of mRNA- including mRNA identity, 5' capping efficiency and 3' poly(A) tail length and heterogeneity and 3) Analysis and characterisation of mRNA multimerisation (aggregation) using non-denaturing IP-RP HPLC.

Characterizing mRNA therapeutics, particularly poly(A) tails, remains challenging due to heterogeneity and the poor performance of conventional sequencing on homopolymers. Direct RNA sequencing overcomes these limitations by reading full-length molecules. In this talk, I will present our analytical strategies for quantifying poly(A) features, which enabled us to uncover unexpected metabolic pathways that stabilize mRNA therapeutics.

RNAi is well-positioned to positively impact the lives of patients living with obesity and related metabolic disorders, due to its strong clinical track record of safety, efficacy and durability. As a modality, mRNA silencing enables pharmacological intervention in diverse disease-associated mechanisms including thermogenesis, lipogenesis, energy expenditure and inflammation. SanegeneBio is developing differentiated tissue-selective delivery technologies using our LEAD (Ligand and Enhancer Assisted Delivery) platform. In this presentation, we will share Phase 1 clinical data demonstrating potential for best-in-class siRNA payloads, our novel ligand-based approach targeting adipocytes and skeletal muscle, and preclinical proof-of-concept for several obesity targets. Together, these data support the promise of RNAi-based medicines as a transformative modality in the treatment of obesity—capable of addressing limitations of existing therapies and establishing a new standard of care.

We developed the FORCE™ platform to enable TfR1-mediated delivery of oligonucleotides and other therapeutic payloads to both muscle and the CNS. The platform combines a monovalent TfR1-binding antibody fragment and a payload specifically designed to target the underlying cause of disease. Its modular nature allows delivery of diverse payloads, each operating through distinct mechanisms of action. Emerging preclinical and clinical data across multiple programs highlight the potential of the FORCE platform to drive functional improvement in serious neuromuscular disorders, including those with neurological involvements.

The GLP-1 boom in obesity and metabolic disease has triggered a capacity crunch, hitting emerging biotechs hardest. This talk explores two pressure points in GLP-1 API manufacturing: (1) defining defensible starting materials for complex dual agonists under regulatory scrutiny, and (2) securing scarce development and GMP capacity at CDMOs dominated by big pharma. We share pragmatic tactics to keep small sponsors competitive without sacrificing quality or speed.v

Over the past decade, the pharmaceutical industry has transitioned from traditional small molecules toward complex new modalities, including peptides, antibody-drug conjugates (ADCs), and oligonucleotides. The structural complexity and similarities inherent in these modalities pose significant characterization challenges, requiring analytical scientists to extend the boundaries of modern techniques. While Size Exclusion Chromatography (SEC) and Reversed-Phase Liquid Chromatography (RPLC) are essential for assessing physical stability and chemical purity, one-dimensional chromatography is often insufficient for full characterization. To address this, we have developed an array of multidimensional techniques coupling orthogonal retention mechanisms, such as RPLC-RPLC, RPLC-SEC, SEC-RPLC, and RPLC-HILIC. A primary challenge in these systems is mobile phase incompatibility between dimensions; we address this through a novel interface utilizing In-Line Mixing Modulation (ILMM) for sample homogenization. Furthermore, non-denaturing SEC methods are critical for peptides to preserve native conformation and higher-order structures (HOS), which are essential for biological activity and quality control. Developing these methods is often difficult due to secondary interactions that limits the use of physiological aqueous buffers requiring reasonable amounts of organics. We present strategies to suppress these interactions, enabling separations based on pure hydrodynamic volume. This presentation will highlight the current analytical challenges and the advanced strategies to achieve comprehensive peptide characterization.

As mRNA modalities expand, traditional analytical approaches can struggle to capture RNA quality and function. Sequencing-based characterization overcomes these limitations by providing high-resolution insight into integrity, impurities, and translation dynamics, enabling drug developers and manufacturers to establish a stronger foundation for mRNA development and process control.

Recently, the regulatory landscape for synthetic oligonucleotides has evolved dramatically with the publication of draft guidelines from EMA and CDE. In addition, oligonucleotides will be in scope of the proposed revisions to ICH Q6. This presentation will explore the emerging trends in these regulations, including areas of harmonization and divergence.

Areas for application of platform data and approaches will be discussed. Experience interacting with health authorities will be presented in addition to potential future activities and interactions.

This work demonstrates that Arrhenius-based accelerated predictive stability (APS) can reliably predict siRNA shelf life. APS complements conventional stability studies, with predictions closely matching real-time data. This enables conservative, data-driven shelf-life assessments, supports informed long-term stability decisions, and accelerates development timelines to help siRNA therapies reach patients faster.

This presentation outlines a risk-based strategy for microbiological control in synthetic oligonucleotide drug substance manufacturing. Key recommendations include facility design, environmental monitoring, equipment cleaning, and in-process controls. Emphasizing proactive risk assessment and best practices, the framework ensures consistent production of high-quality oligonucleotide therapeutics and compliance with evolving regulatory expectations.

Robust analytical and process strategies are essential to ensure the therapeutic quality of oligonucleotides modified with phosphorothioate (PS) groups, which are required for nuclease resistance and enhanced efficacy. This PS modification introduces analytical complexity by creating a chiral center and also complicates control strategy because the drug is commonly dosed as a mixture of all its P-diastereomers. It is possible that different diastereomers have different pharmacological properties and different biological activities. Therefore, it is essential to maintain consistent diastereomeric distribution across drug substance batches to justify the consistent efficacy of the drug. While a consistent diastereomeric distribution is typically achieved by using the same coupling activator in solid-phase oligonucleotide synthesis (SPOS), it has been suggested that chromatographic purification of siRNA single strands might separate these diastereomers, altering their ratios. Additionally, it is a regulatory requirement to assess and describe the comparability of diastereomeric profile to justify any changes to the synthetic process. In developing liquid chromatography methods to separate and quantify diastereomers, we found that diastereomer separation is highly dependent on sequence and physical properties, particularly the propensity of single strands to form higher-order structures. Bespoke analytical methods were required for each oligonucleotide, so at AstraZeneca we developed an approach to efficiently develop methods capable of separating and quantifying 2 to 16+ P diastereomers in diverse single-strands and duplexes of siRNA. We also showed that significant diastereomer separation can occur during chromatographic (SAX) purification under certain conditions, potentially causing variability in diastereomer ratio. Characterization of the molecules linked oligonucleotide structure to a higher risk of separation during purification, and thus to a risk of altering the diastereomer ratio. Accordingly, we propose factors to consider when developing a robust, scalable oligonucleotide manufacturing process and mitigations to reduce the risk of changing the diastereomer profile—applicable to SPOS and biocatalytic processes—to enable a seamless clinical program with no risk of changing pharmacological properties between batches of siRNA. Our work provides a case study in robust analytical and manufacturing practices, supporting sustainable, scalable production of oligonucleotide therapeutics. We highlight critical analytical criteria and process recommendations that facilitate seamless clinical progression and regulatory acceptance, ensuring that pharmacological properties remain consistent throughout development and commercialization.

Chromatographic separation of product-related impurities in antisense oligonucleotides (ASOs) is inherently challenging due to the structure similarity among closely related species. Coupling mass spectrometry (MS) to chromatography and UV detection has been a successful strategy to overcome some of the challenges; however, it does not address the need to implement orthogonal chromatographic methods to ensure comprehensive characterization of ASO impurities through distinct separation techniques. In this study, we developed a two-dimensional liquid chromatography (2D-LC) method for the analysis and quantitation of major product-related impurities in ASOs. The method integrates a hydrophilic interaction chromatography (HILIC) in the first dimension with a weak anion exchange (WAX) separation in the second, thus providing high orthogonality based on differences in polarity and charge. We employed Design of Experiments (DoE) to guide method development and systematically optimized key chromatographic parameters in both dimensions. The final optimized method was configured in selective comprehensive mode, and the main peak region from the first dimension was fully sampled in multiple cuts and analyzed by fast WAX separation. The method demonstrated excellent linearity, sensitivity, and quantitation limits. This fully UV-based 2D-LC platform offers a practical solution for ASO impurity analysis without the need for MS detection and analysis, suitable for both development and routine quality control. The ability to switch between HILIC-WAX and HILIC-MS modes enhances the method’s versatility in oligonucleotide characterization and complex impurity profiling.

Background: Immune-modulating nanobiologics (INBs) are a new class of lipid nanoparticle (LNP)-encapsulated therapeutic mRNAs, designed by integrating viral immune evasion strategies, insights into intracellular pathways driving pathological immune responses, and recent advances in mRNA-based therapeutics. This innovative approach offers a promising treatment modality for the treatment of autoimmune and autoinflammatory (I&I) diseases. Type I interferonopathies—characterized by chronic overexpression of type I interferons (IFN-I) and interferon-stimulated genes (ISGs)—play a central pathogenic role in several I&I conditions. In this study, we investigated the preclinical safety and therapeutic efficacy of INBs specifically engineered to modulate aberrant IFN-I signaling, using a murine model of systemic lupus erythematosus (SLE). Methods: Immunogenicity was evaluated by quantifying serum IFN-β concentrations 3 days after a single IM injection of INB in female C57Bl mice. Efficacy was assessed using the pristane-induced SLE model in female C57Bl mice. Six months after pristane injection, INBs were administered intramuscularly (IM) either once or twice (with a 2-week interval). Disease progression was monitored over 30 days. Serum IFN-β levels and INB protein expression in immune cells were quantified using designated IFN-β and FLAG ELISA kits, respectively. Results: INB3, our lead candidate, was tolerable and showed no signs of immunogenicity. INB3 was detected in circulating white blood cells 3 days post-injection, and in splenocytes and draining lymphocytes by Day 30. Respectively, the pristane-induced overexpression of serum IFN and the high, pathological, relative spleen and kidneys weight were significantly reduced compared to the vehicle-treated controls. The treatment effect was maintained for at least 30 days. Conclusion: INB3 was safe and demonstrated potent inhibition of type I interferon production and significant therapeutic benefit in a murine model of SLE, highlighting its potential as a novel treatment for the treatment of I&I diseases associated with aberrant type I IFNs expression. These preliminary findings warrant further investigation in advanced preclinical models to support the initiation of early-phase clinical trials as a targeted immunomodulatory therapy for SLE and other IFN-driven I&I diseases.

Early clinical data with autologous lentiviral chimeric antigen receptor (CAR) T cell therapy in autoimmune disease have been paradigm shifting with potential to transform patient care. However, traditional CAR-T therapies have limitations on scalability, access, procedure complexity, and safety risks associated with conditioning that may limit their reach. Sail Biomedicines is developing a novel, off-the-shelf intravenous (IV) injectable product to enable in vivo transient programming of a patient's immune cells without the need for lymphodepletion. Methods: SAIL’s in vivo CD19 CAR-T platform comprises two key components: (1) circular, Endless RNA™ (eRNA™) designed for enhanced stability and durable expression of CD19 CAR in T cells, and (2) targeted nanoparticles (TNPs) with optimized surface functionalization and composition to maximize endosomal escape and eRNA expression in T cells. Our eRNA-TNP platform for engineering CAR-T cells was evaluated in vitro using primary human and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and in vivo utilizing humanized mouse models and non-human primates. Results: Leveraging these technology platforms, TNP delivery of eRNA-encoded CD19 CAR payload achieved superior dose potency and a longer expression window than mRNA, generating significantly higher cumulative number of CAR+ T cells over time. SAIL’s eRNA-TNP resulted in efficient depletion of B cells in vitro and was equally potent at engineering T cells in PBMCs from both healthy and systemic lupus erythematosus (SLE) donors. In vivo delivery of eRNA-TNP resulted in efficient and selective expression of the CAR protein in CD4 and CD8 T cells in blood and lymphoid tissues in CD34+ humanized NSG mice and cynomolgus monkeys. Following a short dosing cycle, in vivo-engineered T cells with SAIL’s eRNA-encoded CD19 CAR expressed a robust number of CAR molecules per cell and achieved deeper depletion of B cells than ORF-matched mRNA in both blood and lymphoid tissues; the latter of which is known to be critical to achieve long-lasting immune reset in autoimmune disease patients. Repopulating B cells after depletion exhibited a largely immature phenotype, indicative of effective reset of the B-cell compartment and potential for long-term remission without sustained immunosuppression. Conclusion: SAIL’s eRNA-TNP platform enables in vivo transient programming of T cells with an optimal dose efficiency profile. In vivo CAR engineering with eRNA-TNP has the potential to replace a complex, costly, and genome-integrative cell therapy product with an off-the-shelf and non-integrative IV injectable product that avoids the safety risks associated with lymphoablative conditioning. Plans are in place to rapidly advance a first-generation in vivo eRNA-encoded CAR product to human clinical studies for patients with B-cell driven autoimmune diseases, such as SLE.

Vaccination is crucial for combating respiratory infectious diseases, yet conventional intramuscular vaccines primarily induce systemic immunity with limited mucosal protection. To overcome this, we designed a novel mRNA-lipid nanoparticle platform engineered for targeted mucosal delivery that significantly reduced lung viral loads and elicited strong systemic T cell and antibody responses across species.